1. Field of the Invention
The present invention is related to compounds comprising mannitol or glucitol derivatives which may be used to build up oligomeric compounds. The invention is further related to uses of these oligomeric compounds for hybridization and as probes. In addition, methods for the detection of nucleic acids are disclosed wherein the oligomeric compounds are used.
2. Background of the Invention
In the field of molecular diagnostics, the detection of target nucleic acids with the polymerase chain reaction (PCR) plays an important role. The routine screening of blood banks for the presence of Human Immunodeficiency Virus (HIV), or Hepatitis-B (HBV) or C Virus (HCV) is an example for the large-scale application of PCR-based diagnostics. Automated systems for PCR-based analysis often make use of real-time detection of product amplification during the PCR process. Key to such methods is the use of modified oligonucleotides carrying reporter groups or labels.
In its simplest form, PCR is an in vitro method for the enzymatic synthesis of specific nucleic acid sequences, using two oligonucleotide primers that hybridise to opposite strands and flank the target sequence, that is the region of interest in the target nucleic acid. A repetitive series of reaction steps involving template denaturation, primer annealing, and the extension of the annealed primers by DNA polymerase (DNA: desoxyribonucleic acid) results in the exponential accumulation of a specific fragment whose termini are defined by the 5′ ends of the primers. The detection of DNA amplification products generated by a PCR process can, on the one hand, be accomplished in separate working steps. These may involve the characterisation of amplified fragments with respect to their electrophoretic mobility and/or the analysis of denatured amplification products attached to a solid support using a hybridisation probe.
On the other hand, the detection of DNA amplification products can be done in a so-called “homogeneous” assay system. A “homogeneous” assay system comprises reporter molecules or labels which generate a signal while the target sequence is amplified. An example for a “homogeneous” assay system is the TaqMan® system that has been detailed in U.S. Pat. No. 5,210,015, U.S. Pat. No. 5,804,375 and U.S. Pat. No. 5,487,972. Briefly, the method is based on a double-labelled probe and the 5′-3′ exonuclease activity of Taq DNA polymerase. The probe is complementary to the target sequence to be amplified by the PCR process and is located between the two PCR primers during each polymerisation cycle step. The probe has two fluorescent labels attached to it. One is a reporter dye, such as 6-carboxyfluorescein (FAM), which has its emission spectra quenched by energy transfer due to the spatial proximity of a second fluorescent dye, 6-carboxy-tetramethyl-rhodamine (TAMRA). In the course of each amplification cycle, the Taq DNA polymerase in the process of elongating a primed DNA strand displaces and degrades the annealed probe, the latter due to the intrinsic 5′-3′ exonuclease activity of the polymerase. The mechanism also frees the reporter dye from the quenching activity of TAMRA. As a consequence, the fluorescent activity increases with an increase in cleavage of the probe, which is proportional to the amount of PCR product formed. Accordingly, amplified target sequence is measured detecting the intensity of released fluorescence label.
A similar principle of energy transfer between fluorescent dye molecules applies to “homogeneous” assays using so-called “molecular beacons” (U.S. Pat. No. 6,103,476). These are hairpin-shaped nucleic acid molecules with an internally quenched fluorophore whose fluorescence is restored when they bind to a target nucleic acid (U.S. Pat. No. 6,103,476). They are designed in such a way that the loop portion of the molecule is a probe sequence complementary to a region within the target sequence of the PCR process. The stem is formed by the annealing of complementary arm sequences on the ends of the probe sequence. A fluorescent moiety is attached to the end of one arm and a quenching moiety is attached to the end of the other arm. The stem keeps these two moieties in close proximity to each other, causing the fluorescence of the fluorophore to be quenched by energy transfer. Since the quencher moiety is a non-fluorescent chromophore and emits the energy that it receives from the fluorophore as heat, the probe is unable to fluoresce. When the probe encounters a target molecule, it forms a hybrid that is longer and more stable than the stem hybrid and its rigidity and length preclude the simultaneous existence of the stem hybrid. Thus, the molecular beacon undergoes a spontaneous conformational reorganisation that forces the stem apart, and causes the fluorophore and the quencher to move away from each other, leading to the restoration of fluorescence which can be detected.
More examples for “homogeneous” assay systems are provided by the formats used in the LightCycler® instrument (see e.g. U.S. Pat. No. 6,174,670), some of them sometimes called “kissing probe” formats. Again, the principle is based on two interacting dyes which, however, are characterised in that the emission wavelength of a donor-dye excites an acceptor-dye by fluorescence resonance energy transfer. An exemplified method uses two modified oligonucleotides as hybridisation probes, which hybridise to adjacent internal sequences of the target sequence of the PCR process. The 5′-located modified oligonucleotide has a donor-dye as a label at its 3′ end. The 3′-located modified oligonucleotide has an acceptor-dye at its 5′ end. Following the head-to-tail-oriented annealing of the two modified oligonucleotides to the target sequence in the course of an amplification cycle, donor and acceptor dye are brought in close proximity. Upon specific excitation of the donor dye by means of a monochromatic light pulse, acceptor dye fluorescence is detected providing a measure for the amount of PCR product formed.
The oligomeric compound or modified oligonucleotides used in “homogeneous” assay systems comprise nucleotides or modified nucleotides, i.e. the monomeric units, to which labels such as dyes as reporter molecules are attached. The features of such monomeric units are that they                (1) can be attached to and/or integrated into the sugar-phosphate polymer backbone of a nucleic acid,        (2) do not prevent the pairing of the modified oligonucleotide with its complementary target sequence,        (3) provide functional groups for the attachment of one or more labels.        
In addition, the TaqMan® format requires that the oligomeric compound can be digested by 5′-3′ exonuclease activity of a template-dependent DNA-polymerase.
Several compounds and their use for incorporation as monomeric units into nucleic acids are known in the art. Such compounds provide functional groups and/or linking moieties for the covalent attachment of reporter groups or labels. In the course of the chemical synthesis of the oligomeric compound, the skeletal structure of the “non-nucleotide compound” or “modified nucleotide” is connected with the “oligonucleotide” backbone, for example by phosphoramidite-based chemistry resulting in a phosphodiester. A given incorporated compound thus represents a modified nucleotide within the newly generated “modified oligonucleotide”. A label is bound by a functional group of a linking moiety, exemplified by but not limited to an amino function that is present on the skeletal structure proper or on the “linking moiety”, which connects the skeleton with the functional group. A label can be covalently attached to the compound prior to the synthesis of a “modified oligonucleotide” or afterwards, upon the removal of an optional protecting group from the functional group to which the label is to be coupled.
EP 0135587 describes modifications of conventional nucleosides which carry a reporter group attached to a substituent group of the nucleotide base. EP 0313219 discloses non-nucleoside reagents characterised by a linear hydrocarbon skeletal structure with a linking moiety, or a side group to which a label can be bound. EP 0 313 219 is silent about other types of skeletal structures and their particular properties. U.S. Pat. No. 5,451,463 describes trifunctional non-nucleotide reagents, particularly 1,3-diol-based skeletal structures possessing a primary amino group. Such reagents can be used for example for terminal labelling of 3′ termini of oligonucleotides. WO 97/43451 discloses non-nucleotide reagents based on a carbocyclic (C5 to C7) skeletal structure, whereby a substituted or unsubstituted cyclohexane is preferred. According to the document, such a structure provides rigidity which is necessary to extend a functional moiety, e.g. a functional group to which a reporter group can be coupled, away from the oligomeric backbone of the modified oligonucleotide. This is desired because the coupling efficiency of the reagent after the incorporation into a modified oligonucleotide is enhanced. Sheng-Hui, S., et al., Bioorganic & Medicinal Chem. Lett. 7 (1997) 1639-1644, describe non-nucleotide compounds based on a cyclohexane skeletal structure, particularly on the compound cyclohexyl-4-amino-1,1-dimethanol. The integration into the oligonucleotide backbone is made possible by functional groups substituting the methyl residues at the C1 position. To the amino group a linking moiety is attached which carries a label.
There are also disclosures with regard to glucitol or mannitol-based modified nucleosides. Compounds derived from 1,5-anhydro-2,3-dideoxy-hexitol are known to the art from several documents which, however, are focused on the hexitol compounds per se or on hexitol-based modified nucleosides. Such modified nucleosides can be used as drugs or for the purpose of chemical synthesis, particularly the synthesis of modified oligonucleotides. Pravdic, N., et al., Croatica Chemica Acta 45 (1973) 343-356, describe the synthesis of 1,5-anhydro-2-acetamido-2,3-dideoxy-D-hexitol, i.e. the -mannitol or -glucitol derivative (compound XVIII). The document is, completely silent about particular uses of such compounds, other than for chemical synthesis. JP 60016982 describes the synthesis of 1,5-anhydro-3-deoxy-D-glucitol. The compound is described for the use of suppressing the activity of glucose-acceptive neurons. WO 93/25565, van Aerschot, A., et al., Bioorganic & Medicinal Chemistry Letters (1993) 1013-1018; Verheggen, I., et al., J. Med. Chem. 36 (1993) 2033-2040; Verheggen, I., et al., J. Med. Chem. 38 (1995) 826-835; and Perez-Perez, M.-J., et al., Bioorg. & Med. Chem. Lett. 6 (1996) 1457-1460, describe 1,5-anhydro-2,3-dideoxy-D-hexitol derivatives that carry at the C2 position a hydroxyl residue or a heterocyclic base. Andersen, M. W., et al., Tetrahedron Lett. 37 (1996) 8147-8150 describe similar modified nucleosides; however, the authors also mention 1,5-anhydro-2,3-dideoxy-D-hexitol derivatives that carry at the C2 position a hydroxyl residue or an amino residue. WO 9605213 and Hossain, N., et al., J. Org. Chem. 63 (1998) 1574-1582 describe the synthesis of modified nucleosides derived from 1,5-anhydro-2,3-dideoxy-D-glucitol and -mannitol, respectively. The latter two documents disclose the synthesis of modified oligonucleotides having incorporated hexitol-based modified nucleosides.
Compounds to be used for the incorporation of labels into nucleic acids have to be carefully selected as they may:                (a) interfere with base pairing,        (b) fail to provide a skeletal structure of sufficient rigidity,        (c) provide largely hydrophobic structures resulting in low water solubility,        (d) provide only limited amenability to chemical modifications,        (e) comprise mixtures of enantiomers        
Therefore, it was an object of the present invention to provide new compounds to be used for the incorporation of labels into nucleic acids.